Immunofluorescence

patterns on immunofluorescence. Preparation of fluorescence To perform immunofluorescence staining, a fluorophore must be conjugated ("tagged") to an antibody. Staining procedures can be applied to both retained intracellular expressed antibodies, or to cell surface antigens on living cells. There are two general classes of immunofluorescence techniques: primary (direct) and secondary (indirect). The direct attachment of the fluorophore to the antibody reduces the number of steps in the sample preparation procedure, saving time and reducing non-specific background signal during analysis. ==Limitations==

Immunofluorescence is only limited to fixed (i.e. dead) cells, when studying structures within the cell, as antibodies generally do not penetrate intact cellular or subcellular membranes in living cells, because they are large proteins. To visualize these structures, antigenic material must be fixed firmly on its natural localization inside the cell. To study structures within living cells, in combination with fluorescence, one can utilize recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). The GFP-technique involves altering the genetic information of the cells. A significant problem with immunofluorescence is photobleaching, == Advances ==

The main improvements to immunofluorescence lie in the development of fluorophores and fluorescent microscopes. Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability. of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch–Schönlein purpura: IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis. Super-resolution fluorescence microscopy methods can produce images with a higher resolution than those microscopes imposed by the diffraction limit. This enables the determination of structural details within the cell. Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores (spectral overlap). Some of the recently developed super-resolution fluorescent microscope methods include stimulated emission depletion (STED) microscopy, saturated structured-illumination microscopy (SSIM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). == Notable people ==

• Albert Hewett Coons (1912–1978), physician, pathologist and immunologist • Cornelia Mitchell Downs (1892–1987), microbiologist and journalist == See also ==